Vaccination is an important tool for handling health care programs both in developed and developing nations. The number of recommended vaccines has increased significantly in recent years against individual infections. Presently, the schedule of infants and children may require more than 24-25 separate shots of vaccines for effective immunization against life threatening diseases.
The majority of vaccines currently in development belong to a specific class of subunit vaccine compositions, which consist of recombinant or purified pathogen-specific proteins or encoded antigens (from DNA) that will be expressed and presented in vivo in order to accomplish or elicit the desired immunogenic response. This type of vaccine presents an antigen to the immune system without introducing viral particles, whole or otherwise. While evidence suggests that live, attenuated pathogens and viral vectors can induce protective effects, they often cause unwanted side effects or raise safety concerns, which is one reason why subunit vaccines have risen to prominence in the field (Arvin et al., “New viral vaccines”, Virology, 344:240-249 (2006); Yang et al., “A novel peptide isolated from phage library to substitute a complex system for a vaccine against staphylococci infection”, Vaccine, 24:1117-1123 (2006)).
A combination vaccine which can provide immunogenicity against large number of diseases is always advantageous over the monovalent vaccines. We cannot reduce the number of immunizations required in infants and children to protect them from various fatal diseases but the compliance can be increased by reducing the number of separate vaccinations. A combination vaccine is advantageous over monovalent vaccine as it not only increases the compliance but it is also cost effective and convenient thereby reducing the chances of missing any vaccination. However, due to complications associated with the preparation of such combination vaccines due to possible interaction between the antigens has always been a challenge before the scientific community. Complications include immune interference when vaccine antigens are administered concurrently at the same site in the body, and instability of the antigens together in combinations, impeding the simultaneous development of protective immunity.
Combination vaccines, while greatly desired for ease of administration and increased compliance, pose difficulties in development due to factors including: physical and biochemical incompatibility between antigens and other components, immunological interference and stability. For instance, adjuvants (e.g. aluminum salt, oil-in-water emulsions) that modify the effects of other agents, such as a drug or vaccines, have few if any direct effects when administered alone. Adjuvants are often included in vaccines to enhance a recipient's immune response to a supplied antigen, while keeping the injected foreign material to a minimum. In a combination vaccine, a specific adjuvant may reduce the activity of one antigen and excessively increase the reactivity of another antigen. Buffers used to minimize changes in acidity of a solution (when an acid or base is added) may also interact with other vaccine components. Stabilizers are used to ensure vaccines maintain effectiveness during storage by counteracting the effects of temperature, pH and preservatives. Vaccine stability is essential, particularly where the cold chain is unreliable. However, stabilizers may also have an effect on other agents present in the vaccine. All these potential interactions between vaccine components can negatively effective vaccine potency reducing the immunogenicity of the vaccine.
The trend towards combination vaccines has the advantage of reducing discomfort to the recipient, facilitating scheduling, and ensuring completion of regiment. However, there is also the concomitant risk of reducing the vaccine's efficacy. It would be, therefore, advantageous to make vaccine combinations which meet the needs of a population, and which, in addition, do not interfere with each other. It would be of further advantage if the combination of the vaccines results in synergy with resulting improvement of one or both vaccines' efficacy, or improved correlates of protections for one or both vaccines.
An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly distinguished into two extreme categories, being humoral or cell mediated immune responses (traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Th2-type immune responses (humoral response) and Th1-type responses (cell-mediated response).
Extreme Th1-type immune responses may be characterized by the generation of antigen specific, haplotype-restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice, Th1-type responses are often characterized by the generation of antibodies of the IgG2a subtype, while in the human these correspond to IgG1 type antibodies. Th2-type immune responses, on the other hand, are characterized by the generation of a broad range of immunoglobulin isotypes, including (in mice) IgG1, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines, a number of identified protein messengers which serve to help the cells of the immune system and steer the eventual immune response to either a Th1 or Th2 response. Thus, high levels of Th1-type cytokines tend to favor the induction of cell mediated immune responses to the given antigen, while high levels of Th2-type cytokines tend to result in the induction of humoral immune responses to the antigen.
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either Th1- or Th2-type cytokine responses. Traditionally the best indicators of the Th1:Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Th1 or Th2 cytokines by T lymphocytes in vitro after re-stimulation with antigen, and/or the measurement of the IgG1:IgG2a ratio of antigen specific antibody responses.
Thus, a Th1-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Th1-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Th1-type isotype. Suitable adjuvant systems which promote a predominantly Th1 response include Monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A, and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminum salt.
Adjuvants are molecules, compounds, or macromolecular complexes that boost the potency and longevity of specific immune response to antigens, but cause minimal toxicity or long-lasting immune effects on their own. Adjuvants can be used to enhance immunogenicity, modulate the type of immune response, reduce the amount of antigen or the number of immunizations required, and improve the efficacy of vaccines in newborns or elderly. To be maximally effective, adjuvants must be selected judiciously and formulated appropriately based on the desired immune response. However, the number of adjuvants with acceptable efficacy and safety profiles is limited, and these proprietary molecules/compounds are in the hands of a few companies, as is most of the formulation expertise.
Vaccines containing recombinant proteins require an adjuvant to elicit a durable immune response (Callahan, Shorter, et al., 1991, The importance of surface charge in the optimization of antigen-adjuvant interactions, Pharm Res, v 8:851-8). The default position in developing subunit protein immunogens for human vaccines is to utilize aluminum adjuvants as the starting point. The use of aluminum adjuvants is thus fostered by the fact that the record of safety of newer formulations cannot match the long term acceptability of aluminum adjuvants in human vaccines. Overall, this has amounted to a lack of advanced adjuvants that can be applied to vaccine development, coupled with the fact that several of the most advanced adjuvant formulations/compounds are the property of large pharmaceutical companies. Aluminum-salt adjuvants are currently the most widely used adjuvants for general use in humans. Aluminum adjuvants are considered relatively weak, effective in generation of neutralizing antibodies against certain bacterial antigens, but relatively ineffective at inducing cellular immune responses. There is some consensus that the more effective vaccines with aluminum are those in which antigen is bound to the aluminum surface, rather than free in solution (Lindblad, 2004, Aluminium adjuvants—in retrospect and prospect, Vaccine, v 22:3658-68). For reproducibility of formulations and stability, it is desirable to define conditions for optimal binding of antigen to crystal surfaces, and conditions in which antigen does not desorb over time or under elevated stress conditions. To construct aluminum vaccines, it is necessary to carry out studies to optimize binding and desorption. Aluminum adjuvants have a point of zero charge (PZC) at a certain solution pH, but are charged at pHs above or below this value (White and Hem, 2000, Characterization of aluminium-containing adjuvants, Dev Biol (Basel), v 103:217-28). Selecting an optimal formulation pH is further complicated for a recombinant protein vaccine for which binding to aluminum salt adjuvants is generally required to obtain the desired immune response (McInerney, Brennan, et al., 1999, Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide, Vaccine, v 17:1359-68). To facilitate protein binding to adjuvant, a solution pH is selected in which the protein and adjuvant have opposite charges. However, a solution pH that provides optimal protein stability, may not allow for appropriate binding of the vaccine to adjuvants. In such a scenario, a vaccine protein may have to be prepared at pH that is suboptimal for stability and lyophilized with appropriate stabilizing excipients to minimize degradation during long-term storage.
The emerging trend in these fields has been that it is not sufficient to engineer the protein target itself, but that potent, safe, adjuvant formulations must be utilized as an intrinsic component of vaccine design, from the earliest feasibility experiments through clinical testing. The use of formulation technology can result in a significant decrease in dose levels and number of vaccinations, an increase in the quality and breadth of the immune response, as well as long-term, sustained responses to the antigenic target. Adjuvant formulations consist of aqueous suspensions of vaccine particles, adsorption of antigens to salts of aluminum, oil-in-water emulsions of antigens, and nanovesicles such as liposomes or niosomes.
In the case of aluminum adjuvants, it has been suggested that to provide adequate immunogenicity, antigens must be adsorbed on the surface of the adjuvant. (Gupta et al., 1995, Adjuvant Properties of Aluminum and Calcium Compounds, Pharmaceutical Biotechnology, 6: 229-248; and White and Hem, 2000, Characterization of aluminum-containing adjuvants, Dev Biol (Basel), 103: 217-28). This adsorption is typically facilitated through electrostatic interactions between the antigen and adjuvant, and the formulation pH is usually chosen so that the antigen and adjuvant are oppositely charged (Callahan et al. 1991). The surface charge on the adjuvant also can be modified by surface exchange reactions with buffer salts such as phosphate, succinate, and citrate (Hem and White, 1984, Characterization of aluminum hydroxide for use as an adjuvant in parenteral vaccines. J Parenter Sci Technol, 38(1): p. 2-10; Chang et al., 1997, Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. PDA J Pharm Sci Technol, 51(1): p. 25-9; and Rinella et al., 1996, Treatment of aluminium hydroxide adjuvant to optimize the adsorption of basic proteins. Vaccine, 14(4): p. 298-300.)
The mechanisms of action of aluminum-salt adjuvants are poorly understood, but are likely due to several different mechanisms. (Lindblad 2004. “Aluminium compounds for use in vaccines” Immunol. Cell Biol, 82(5):497-505; Gupta and Siber, 1995, Adjuvants for Human Vaccines—Current Status, Problems and Future-Prospects. Vaccine 13(14):1263-1276; Gupta and Rost, 2000, Aluminum Compounds as Vaccine Adjuvants, In O'Hagan D, editor Vaccine Adjuvants: Preparation Methods and Research Protocols, ed., Totowa, N.J.: Humana Press Inc. p 65-89; Cox and Coulter, 1997, Adjuvants—a classification and review of their modes of action, Vaccine 15(3):248-256). Common proposed mechanisms are that the adjuvant acts as a depot at the site of injection, wherein the antigen is slowly released after administration. (Cox and Coulter, 1997)).
Another proposed mechanism is that the adjuvant aids in delivery of the antigen to antigen-presenting cells (Lindblad 2004). A further proposed mechanism is that adjuvant serves as an immunostimulator and elicits Th2 cytokines (Grun and Maurer 1989, Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses, Cell Immunol, 121(1):134-145)). Yet another proposed mechanism is that the presence of an adjuvant destabilizes protein antigens on the surface of the adjuvant making them more susceptible to proteolytic degradation (Jones et al., 2005, Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens. J Biol Chem 280(14):13406-13414; and That et al., 2004. “Antigen stability controls antigen presentation” J Biol Chem, 279(48):50257-50266)).
Although the mechanism of action is not fully understood, it is likely that surface area, surface charge, and morphology of the adjuvant are important factors dictating the immune response to antigens adsorbed onto these adjuvants (Hem and White 1984). It is generally theorized that the smaller the particle size of the vaccine adjuvant, the more immunogenic the vaccine preparation, especially when particle size is approximately 1 micron, a size best suited for uptake into professional antigen presenting cells (Maa et al., 2003. Stabilization of alum-adjuvanted vaccine dry powder formulations: mechanism and application. J Pharm Sci 92(2):319-332, Diminsky et al., 1999. Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles. Vaccine 18(1-2):3-17).
Aluminum-salt adjuvants provide a well explored means to augment the immunogenicity of protein or peptide subunit vaccines. However, a variety of exploratory formulations to enhance vaccines have been developed as more potent alternative to aluminum-salts adjuvants, but are not currently available in FDA-licensed human vaccines. Formulations designed to enhance immune responses include a variety of compositions based on water-in-oil emulsions, oil-in-water emulsions, self-assembling macrostructures, cytokines, saponins, toll-like receptor agonists (TLR-4, TLR-5, and TLR-9), immunostimulatory double stranded RNA species, unmethylated DNA oligonucleotides, and polymeric microparticles and nanostructures. Many of these compositions are directed towards improving the immunogenicity of injected vaccines, and some variations can be applied to altering routes of delivery for intranasal or oral vaccination.
As an example of one class of immunostimulatory molecules that can be used to enhance vaccine immunogenicity, bacterial DNA, but not vertebrate DNA, can be used because of direct immunostimulatory effects that activate lymphocytes. This is due to unmethylated CpG dinucleotides being present at the expected frequency in bacterial DNA but are under-represented and methylated in vertebrate DNA (Krieg et al., 1995). Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain an unmethylated CpG dinucleotide in a particular sequence context. CpG DNA induces proliferation of almost all (>95%) B cells and increases immunoglobulin (Ig) secretion. This B cell activation by CpG DNA is T cell independent and antigen non-specific. However, B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995). This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG DNA promotes antigen specific immune responses.
In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12 (Klinman et al., 1996; Halpern et al., 1996; Cowdery et al., 1996). These cytokines stimulate natural killer (NK) cells to secrete gamma-interferon (IFN.gamma.) and have increased lytic activity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces a Th1-like pattern of cytokine production dominated by IL-12 and IFN-gamma with little secretion of Th2 cytokines (Klinman et al., 1996).
Other molecules stimulate toll like receptors. One example is flagellin, the protein subunit comprising numerous bacterial flagella. Flagellin is a TLR-5 ligand and triggers at least one of the biological functions of antigen presenting cells upon such binding. Flagella are found on the surface of rod and spiral shaped bacteria, including members of the genera Escherichia, Salmonella, Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema, Legionella, Clostridia, and Caulobacter. The conserved regions of flagellins are important for TLRS binding, while the polymorphic central region can be deleted without affecting binding to TLRS. Flagellin sequences from numerous bacteria are available in the art, such as Genbank accession numbers D13689, YP.sub.-275549, YP.sub.-275550, AAU18718, AAU18717, ZP.sub.-00743095, EA052626, YP.sub.-315348, AAT28337, AAT28336, AAT28335, AAT28334, AAT28333, AAZ36356, AAZ33167, AAZ94424, AAZ91670, NP.sub.-414908, BAD18052, and BAD18051.
As a third example of purified adjuvant immune stimulants, nontoxic (chemically synthesized or enzymatically modified) derivatives of gram negative lipopolysaccharides are potent adjuvants and act by stimulating lymphocytes through TLR-4 binding and activation. For example, monophosphoryl lipid A (MPL) is a derivative of the lipid A component of lipopolysaccharide and is a potent activator of pro-inflammatory cytokines. Although native lipid A and its parent LPS have powerful pyrogenic properties and in humans induce febrile responses (Greisman and Homick, J Immunol, 109:1210-1215 (1972); Greisman and Homick, J Infect Dis, 128:257-263 (1973); Abernathy and Spink, J Clin Invest, 37:219-225 (1958); Rietschel et al, supra; and Raetz, supra (1993)), MPL and its chemically synthesized analogues are not toxic but induce a compendium of host proinflammatory cytokines including IL-1, IL-6, and TNF-alpha.
In addition, to enhance the immune response to subunits adsorbed to aluminum salts, it is likely that co-adjuvants will be required in order to generate effective antibody responses in humans after one or two doses. A number of adjuvant compounds that are compatible with aluminum salts have been evaluated as adjuvants in recent years. Primarily these adjuvants include Monophosphoryl Lipid A (MPL) and QS-21, and CpG sequences.
Recent data with anthrax vaccine indicates in human studies that AVA, an AIOH adsorbed vaccine, can be significantly enhanced by adding CpG 7909 to the adjuvant formulations in non-human primates and humans, in terms of total anti-rPA antibodies and anthrax toxin neutralizing antibodies, although no data describe the long term thermal stability of CpG-containing vaccines (Klinman, 2006, CpG oligonucleotides accelerate and boost the immune response elicited by AVA, the licensed anthrax vaccine, Expert Rev Vaccines, 5:365-9).
MPL and QS-21 have been also used with aluminum salts as well as in proprietary oil emulsion formulations being developed by Glaxo Smith Kline Biologics. QS-21 has been evaluated in AlOH vaccines in humans and animal models with good evidence of tolerability and systemic safety. QS-21 is thought to bind to aluminum salts through ionic and hydrophobic interactions, as well as some part of it remaining in solution (in aqueous vaccines) in a micellar form. QS-21 is a saponin purified from tree bark with broad adjuvant effects to induce both antibody and cell mediated immunity. Though the mechanism is not understood, dose levels effective in conjunction with human vaccines have been evaluated.
QS-21 with aluminum has been evaluated in clinical studies and independent safety studies of QS-21 formulated with antigens have been studied. QS-21 has been associated with stinging at the site of injection (that resolves), with very little evidence of systemic side effects (Waite, Jacobson et al., 2001, Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21, Vaccine, 19:3957-67). Several studies in humans have shown that QS-21 enhances responses to antigens that are adsorbed to aluminum. These include several trials in malaria vaccine candidates (Nardin, Oliveira et al., 2000, Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccinees of defined HLA genotypes, J Infect Dis, 182:1486-96; Kashala, Amador et al., 2002, Safety, tolerability and immunogenicity of new formulations of the Plasmodium falciparum malaria peptide vaccine SPf66 combined with the immunological adjuvant QS-21, Vaccine, 20:2263-77), HIV gp120 (Evans, McElrath et al., 2001, QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans, Vaccine, 19:2080-91) and more recently Rhesus macaque trials of Dengue virus subunits in which neutralizing titers and protection were enhanced by QS-21 (Putnak, Coller et al., 2005, An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model, Vaccine, 23:4442-52). The solution stability of QS-21 has been well studied under long term stability studies, and has shown that adjuvant active QS-21 (which actually consists of two isomeric forms) is highly stable in slightly acidic buffers for over 4 years, though less than 10 days at 40° C. (Kensil and Kammer, 1998, QS-21: a water-soluble triterpene glycoside adjuvant, Expert Opin Investig Drugs, 7:1475-82). QS-21 is stored as a dried powder and in that form is stable indefinitely.
Lyophilization of proteins to stabilize structure and activity for storage and reconstitution has been commonly applied to recombinant protein therapeutic proteins. This has been usually accomplished by freeze drying in the presence of disaccharides such as trehalose and other excipients that promote a glass state during process and storage. Proteins can be stored for long term as long as the product is stored below its glass transition temperature (Tg), above which the material transitions into a rubbery state. Excipients are thought to stabilize protein in the amorphous state through interactions of the stabilizer with specific sites substituting for water during drying and by simultaneously suppressing translational and rotational motions of the protein molecule (α-relaxations) or portions of the molecule (β-relaxations).
Drying technology has been less frequently applied to long term storage of vaccines, especially in the case of vaccines adsorbed to aluminum phosphate or aluminum hydroxide adjuvants. Very little data is available on the storage of dried vaccines under elevated temperature conditions, as most of the attempts to generate dried vaccines have been to obtain inhalable powders or preparations able to survive moderate excursions in temperature. For example, because the yellow fever vaccine is used primarily in tropical climates, lyophilization in the presence of stabilizers (lactose, sorbitol) has been used to preserve viability of the live virus vaccine (Monath, 1996, Stability of yellow fever vaccine, Dev Biol Stand, 87:219-25). Without excipients during lyophilization and storage, activity is rapidly lost above −20° C., but the stabilized vaccine can withstand more than two weeks at 37° C. A lyophilized dried vaccine for the cattle disease rinderpest has also been developed and can be employed for up to a month after leaving the cold chain in African field conditions (House and Mariner, 1996, Stabilization of rinderpest vaccine by modification of the lyophilization process, Dev Biol Stand, 87:235-44). Similar attempts to use variations on process and drying have been recently applied to measles vaccine development (Burger, Cape et al., 2008, Stabilizing formulations for inhalable powders of live-attenuated measles virus vaccine, J Aerosol Med Pulm Drug Deliv, 21:25-34; Burger, Cape et al., 2008, Stabilizing Formulations for Inhalable Powders of Live-Attenuated Measles Virus Vaccine, J Aerosol Med) and for dried vaccine powders for influenza where it is likely very important to devise conditions that permit retention of the structure of the immunogen (Amorij, Meulenaar et al., 2007, Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze-drying, Vaccine, 25:6447-57; Amorij, Huckriede et al., 2008, Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities, Pharm Res).
As a stabilizer, small amounts of formaldehyde are occasionally added to vaccines, including the current AVA anthrax vaccine (Biothrax®), and may act by cross-linking proteins forming more immunogenic protein aggregates on the surface of aluminum crystals (Little, Ivins et al., 2007, Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine, Vaccine, 25:2771-7). Formaldehyde had been used historically as the stabilizer of choice in the older vaccines derived from culture supernatants, such as tetanus toxoid, botulinum toxoids, and others. The current AVA vaccine is labeled for 3 year stability, where stability is a function of a number of biochemical evaluations and potency. Although a moderate amount of stability can be achieved with liquid suspension vaccines, it is not likely that all stability parameters can be met for longer storage periods that are required for vaccines to be stockpiled and distributed.
Successful drying of therapeutic proteins, while retaining structure and function, is dependent on the knowledge of the degradation pathways that occur in solution, which can be retarded or eliminated by appropriate drying and excipients for stabilization. For vaccines, function is largely determined by immunogenicity and protection studies, rather than enzymatic activity. In the case of protein immunogens that are adsorbed to aluminum adjuvant crystals, the measurement of function and other parameters in vitro is correspondingly more difficult, since protein may be sequestered and difficult to remove for analysis. Thus, function can only be tested by immunogenicity and protection studies. The tertiary conformation of proteins can affect enzymatic functions, if present, but also can affect immunogenicity of B cell epitopes dependent on conformation. Linear B and T cell epitopes contained therein can be also affected by oxidation (of methionine and cysteine residues) and deamidation (especially of asparagine residues).
pH is one of the most critical formulation variables governing stability of therapeutic proteins. (Carpenter, Chang et al., 2002, Rational design of stable lyophilized protein formulations: theory and practice, Pharm Biotechnol, 13:109-33; Chi, Krishnan et al., 2003, Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation, Pharm Res, 20:1325-36) By affecting the conformational and colloidal stability of proteins in solution, pH can greatly modulate their aggregation rates (Chi, Krishnan et al., 2003, Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation, Pharm Res, 20:1325-36). In addition, rates of deamidation are strongly dependent on pH (Manning, Patel et al., 1989, Stability of protein pharmaceuticals, Pharm Res, 6:903-18). There can be different optimal pH values for physical and chemical stability for a given protein (Kolvenbach, Narhi et al., 1997, Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH2, J Pept Res, 50:310-8). For example, physical stability may be optimal at a pH where deamidation is unacceptably rapid (Chang, Reeder et al., 1996, Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist, Pharm Res, 13:243-9). In such cases, development of a lyophilized formulation where the rates of these reactions are minimized may provide a viable strategy to obtain a stable product. The few published studies examining effects of pre-lyophilization solution pH on the stability of therapeutic proteins during lyophilization and storage in dried formulations demonstrate the importance of this parameter (Prestrelski, Pikal et al., 1995, Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy, Pharm Res, 12:1250-9; Chang, Reeder et al., 1996, Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist, Pharm Res, 13:243-9; Katayama, Kirchhoff et al., 2004, Retrospective statistical analysis of lyophilized protein formulations of progenipoietin using PLS: determination of the critical parameters for long-term storage stability, J Pharm Sci, 93:2609-23). These studies demonstrated the difficulty in identifying a pre-lyophilization solution pH that confers adequate physical and chemical stability to the proteins studied during lyophilization and storage. However, degradation of proteins could be minimized if sufficient amounts of stabilizing excipients are included in the formulation. For example, when recombinant human interleukin-1-receptor antagonist (rhIL-1ra) was formulated prior to lyophilization in a solution containing suboptimal sucrose at levels less than 0.3 mass ratio of sucrose/protein and at pH less than 6.5, severe protein aggregation occurred after lyophilization, during storage and reconstitution (Chang, Reeder et al., 1996, Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist, Pharm Res, 13:243-9). Protein aggregation was minimized following lyophilization from a solution at pH greater than 6, although, deamidation occurred at an unacceptably high rate. Following lyophilization from a solution containing amounts of sucrose greater than 0.3 sucrose/protein mass ratio at pH 6.5, both destabilization pathways could be inhibited. In another example, interleukin-2 (IL-2) had significantly greater structural perturbation during freeze-drying at pH 7, which resulted in higher levels of aggregation after storage and rehydration than samples lyophilized from solutions at pH 5 (Prestrelski, Pikal et al., 1995, Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy, Pharm Res, 12:1250-9). The addition of sucrose to the pre-lyophilization solution formulation at pH 7 improved the stability of IL-2 during storage following lyophilization. More recently, this approach to preformulation has been taken with anthrax rPA to create a dried powder vaccine candidate for nasal administration (Jiang, Joshi et al., 2006, Anthrax vaccine powder formulations for nasal mucosal delivery, J Pharm Sci, 95:80-96). In this case, conditions for optimizing pH and excipient stabilizers were established for rPA in solution prior to lyophilization. As trehalose was one of the excipients determined to stabilize soluble rPA to thermal stress, there was evidence of at least 30 days stability at 40° C. for the dried trehalose-containing vaccines in terms of the total content of rPA in comparison to liquid samples in which rPA quickly disappeared. In an effort to obtain a dried powder composition for epidermal delivery using a gas-driven injection device, it was found that rapid freezing of aluminum-adsorbed hepatitis B vaccine (HBsAg) in the presence of a mixture of mannitol, glycine, and dextran (not more than ˜6% w/v of total excipients) resulted in vaccines that retained particle size and relative immunogenicity in mice after a rapid freezing (spray freeze drying) that involved injection of the sprayed vaccine into liquid nitrogen prior to drying (Maa, Zhao et al., 2003, Stabilization of alum-adjuvanted vaccine dry powder formulations: mechanism and application, J Pharm Sci, 92:319-32). The behavior of the spray-freeze dried vaccines under thermal stress conditions was not determined, although normally lyophilized vaccine aggregated after processing and was minimally immunogenic. Diminished immunogenicity was associated with aluminum particle aggregation after reconstitution.
U.S. Pat. No. 6,890,512 (“Roser et al.”) teaches lyophilization methods that will prevent aggregation of aluminum particles and further discloses a method of preventing gross aggregation during dehydration and rehydration of particulates in suspension by adding to a particulate suspension of aluminum hydroxide in excess of 15% (w/v) of trehalose. Trehalose, alpha.-D-glucopyranosyl-alpha-D-glucopyranoside, is a naturally occurring disaccharide responsible for protection of plant cells from desiccation. Trehalose has been shown to prevent denaturation of proteins during desiccation by forming sugar glasses that immobilize protein structure. However, Roser et al., while disclosing prevention of gross particle aggregation, did not disclose the importance of freezing rate of a particulate suspension or other factors critical to control and maintain pre-lyophilization particle size and protein structure in an aluminum-salts containing vaccine in the presence of trehalose. Maintenance of particle size is a critical parameter in controlling the degree of adsorption of protein immunogens to the surface of aluminum particles, and is influenced by several factors during lyophilization cycle in addition to the content of trehalose or other glassifying excipients. These factors influence the immunogenicity and generation of protective immune responses.
Multivalent vaccine compositions are known in the art and have been described in the literature. WO1993/024148 discloses an invention of multivalent vaccine containing antigens IPV-DPT-Hib-Hepatitis B wherein DPT is adsorbed to AIOH or aluminum phosphate and Hib is adsorbed to only aluminum phosphate, wherein the Hib antigen is used extemporaneously by mixing to the other antigens just prior to the administration.
WO1997/00697 discloses a DPT-Hib and pertussis multivalent vaccine adsorbed to aluminum phosphate, in which one container has a freeze-dried vaccine and the other container comprises a second antigen.
WO1998/000167 discloses a DTaP—IPV-Hib antigen vaccine and WO1999/13906 describes a multiple component vaccine in which certain components may be reconstituted from a lyophilized state by the other components of the vaccine, or may exist in a single solution, and administers the vaccine in a specially designed container at the time when the vaccination is performed.
WO2000/07623 describes a multi-component vaccine composition having acellular pertussis vaccine components (PT and FHA), diphtheria toxoid (DT), tetanus toxoid (TT), a conjugate of a capsular polysaccharide of Haemophilus influenzae type b and tetanus toxoid or diphtheria toxoid (Hib), Hepatitis B Surface Ag (HBsAg) and inactivated poliovirus (IPV) which may be in a single solution, or certain components may be reconstituted from a lyophilized state by the other components of the vaccine.
WO2002/000249 discloses a capsular polysaccharide of Haemophilus influenza type b not adsorbed onto an aluminum adjuvant salt, and two or more further bacterial polysaccharides which may include whole cell pertussis, tetanus toxoid, diphtheria toxoid, Hepatitis B surface antigen (HbsAg), and/or conjugate polysaccharides of N. meningitides type A, or B, or C as antigens in a single quadrivalent and/or trivalent vaccine.
WO2006/097851 discloses a multivalent vaccine which can be prepared extemporaneously at the time of use by mixing together two components the first component comprising D, T, wP and HBsAg antigens and a second component comprising a Hib conjugate and one or more meningococcal conjugates.
WO2007/054820 relates to a vaccine composition wherein the D, T, and aP antigens are specifically adsorbed on aluminum hydroxide and the Hib and the Hep B antigens are adsorbed onto aluminum phosphate which do not exist in a fully liquid stable composition.
WO2008/044611 discloses a method for the preparation of a mixed IPV-DPT vaccine comprising an inactivated poliovirus Sabin strains type I, II, and III grown in Vero cells, a protective antigen against Bordetella pertussis, a diphtheria toxoid and a tetanus toxoid, which involves the step of producing a poliovirus Sabin strain having a high titer.
In the case of lyophilization, all the patent applications mentioned above require reconstitution of one or more of the individual components prior to administration of the vaccine. The mixing of antigenic components prior to administration from different vials results in the diminishment of the antibody titer against any particular antigen owing to interference of antigenic interactions. US Publication No. 2011/0206726A1 teaches a fully liquid hexavalent combination vaccine comprising D-T-aP-IPV, Hep B and Hib antigens wherein Hib is not substantially adsorbed to any adjuvant and D-T-aP adsorbed to AIOH and Hep B adsorbed to aluminum phosphate in a single vial. It also specifies that, antigenic competition in such multivalent vaccines often results in a diminished response to certain individual antigens. The patent application recognizes an inherent problem of mixing of lyophilized vaccines with liquid vaccines which represents supplementary constraint for the practitioner and involves a possibility of the same being carried out in a poor fashion. It also recognizes that the usage of multi-compartment syringes having separate chambers for liquid and lyophilized vaccines is too difficult to execute and no reduction in production costs (and by extension, no reduction of the cost of immunization) of the vaccine is possible.
The prior art is silent with respect to a combination vaccine comprising at least one antigen comprising a ribosome inactivating protein and at least one antigen comprising a toxin derived from bacterial spores.
Ricin is a plant toxin that is classified as a ribosome inactivating protein (RIP) and is a potent member of the AB family of toxins. Ricin is a highly toxic, naturally occurring lectin (a carbohydrate-binding protein) produced in the seeds of the castor plant Ricinus communis. Ricin is thought to be a bioterror threat because of its stability and high potency as well as the large worldwide reservoir created as a by-product of castor oil production. Exposure to ricin results in local tissue necrosis, and general organ failure leading to death within several days of exposure. A dose of purified ricin powder the size of a few grains of table salt can kill an adult human. The median lethal dose (LD50) of ricin if given by injection is around 22 micrograms per kilogram of body weight (1.78 mg for an average adult, around 1/228 of a standard aspirin tablet/0.4 g gross) in humans if exposure is from injection or inhalation. Ricin is toxic by all routes of exposure, but is especially toxic by the aerosol route, resulting in necrosis of lung epithelia within hours of exposure, multifocal hemorrhagic edema and death within 24-36 hours, with an estimated aerosol LD50 of 5-8 micrograms per kilogram of body weight (0.4 mg-0.64 mgs for an average adult).
The enzymatic A subunit (RTA) is an RNA-N-glycosidase which cleaves a specific adenine residue with eukaryotic 28S ribosomal RNA, leading to protein synthesis arrest and cell death. Depurination of this residue results in an immediate cessation of ribosome progression, which subsequently inhibits protein synthesis. Connected to RTA via a single disulfide bond, the B subunit (RTB) mediates binding to its cell surface receptor, terminal galactose and N-acetyl galactosamine moieties on glycoproteins and glycolipids. The ricin toxin B subunit (RTB) binds with micromolar affinity to α(1-3)-linked galactose and N60 acetylgalactosamine residues that are expressed on the surface of all mammalian cell types. Binding of RTB to these receptors mediates internalization and retrograde transport of the ricin holotoxin to the endoplasmic reticulum (ER). In the ER, RTA dissociates from RTB and is retrotranslocated across the ER membrane into the cytosol where it gains access to rRNA targets. Ricin is therefore extremely potent, as it is able to internalize into almost all mammalian cell types. In addition to ribosome inactivating properties, ricin also elicits vascular leak syndrome (VLS), which primarily affects endothelial cells.
Antibodies to ricin, and more specifically the A chain of ricin, can prevent morbidity and mortality, if given passively prior to or very shortly after exposure, but the therapeutic window of opportunity post exposure is likely to be hours, prior to irreversible toxicity, lowering the probability of successful and effective post-exposure intervention. Data based on murine monoclonal antibodies to ricin A chain have indicated that antibodies that neutralize ricin toxin in vitro by inhibiting cytotoxicity of ricin on cultured cells can confer protection to mice by passive transfer. Equally important, non-neutralizing monoclonal antibodies do not confer protection. From these studies, it is inferred that a critical correlate of immunity that must be established in advanced animal models as well as human safety and immunogenicity studies is the capacity of a ricin vaccine to induce ricin neutralizing antibodies, in systemic circulation or located at mucosal surfaces. Further analysis have strongly suggests that the minority of the response to the ricin A chain results in neutralizing antibodies, and further that the neutralizing response is primarily against conformational determinants (O'Hara, Neal, et al., 2010, Folding domains within the ricin toxin A subunit as targets of protective antibodies, Vaccine, v 28:7035-46).
Analysis of mouse, rabbit and macaque sera has led to the identification of at least six immunodominant regions on RiVax™ comprising individual neutralizing and non neutralizing linear epitopes. Analysis available in the literature has also indicated human antibodies react with peptides spanning residues 41-90 (immunodominant region II) and 161-175 (immunodominant region IV), revealing some degree of common epitope recognition across species (Tommasi, Castelletti, et al., 2001, Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones, Clinical and Experimental Immunology, v 125:391-400; Castelletti, Fracasso, et al., 2004, A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin, Clinical and Experimental Immunology, v 136:365-72). Immunodominant region II is located within folding domain I and contains a solvent-exposed α-helix (residues N97-F108) (O'Hara, Neal, et al., 2010, Folding domains within the ricin toxin A subunit as targets of protective antibodies, Vaccine), a target of the protective mAb R70 also known as Univax 70 (Lebeda and Olson, 1999, Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins, Int J Biol Macromol, v 24:19-26). Residues N97-F108 likely constitutes one of the most immunodominant regions on RTA. Immunodominant region IV on RTA spans amino acids I170-T190. There are at least two linear B-cell epitopes within this region. Residues L161 to I175, in particular, were identified as being a conserved target of serum Abs from Hodgkin's lymphoma patients who had been treated with deglycosylated RTA (RTA.dg) immunotoxin (Castelletti, Fracasso, et al., 2004, A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin, Clin. Exp. Immunol, v 136:365-72). A murine IgG1 mAb GD12 was effective in protecting mice against the effects of intraperitoneal and intragastric ricin challenges, thereby establishing that preexisting serum Abs directed against residues in immunodominant region IV are sufficient to confer both systemic and mucosal immunity to ricin, at least in rodents (Neal, O'Hara, et al., 2010, A monoclonal immunoglobulin G antibody directed against an immunodominant linear epitope on the ricin A chain confers systemic and mucosal immunity to ricin, Infect Immun, v 78:552-61). The GD12 epitope is situated within α-helix E, which runs through the core of RTA's domain II and which terminates with two residues (Glu177 and Arg180) that are involved in RTA catalytic activity (O'Hara, Neal, McCarthy, Kasten-Jolly, Brey and Mantis, 2010, Folding domains within the ricin toxin A subunit as targets of protective antibodies, Vaccine; Katzin, Collins, et al., 1991, Structure of ricin A-chain at 2.5 A, Proteins., v 10:251-9; Li, Chiou, et al., 2009, A two-step binding model proposed for the electrostatic interactions of ricin a chain with ribosomes, Biochemistry, v 48:3853-63).
There is a general consensus that protection against gastrointestinal or aerosol exposure to ricin requires antibodies in respiratory or gastrointestinal secretions that effectively intercept ricin before it can adversely affect epithelial tissue (Griffiths, Bailey, et al., 1997, Liposomally-encapsulated ricin toxoid vaccine delivered intratracheally elicits a good immune response and protects against a lethal pulmonary dose of ricin toxin, Vaccine, v 15:1933-9; Griffiths, Bailey, et al., 1998, Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations, Vaccine, v 16:530-5; Griffiths, Phillips, et al., 1999, Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation, Vaccine, v 17:2562-8; Poli, Rivera, et al., 1996, Aerosolized specific antibody protects mice from lung injury associated with aerosolized ricin exposure, Toxicon, v 34:1037-44; Yan, Rill, et al., 1996, Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with ricin toxoid vaccine encapsulated in polymeric microspheres, Vaccine, v 14:1031-8.). In rodents (Griffiths, Phillips and Bailey, 1999, Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation, Vaccine, v 17:2562-8; Brown and White, 1997, Ultrastructure of rat lung following inhalation of ricin aerosol, International Journal of Experimental Pathology, v 78:267-76; DaSilva, Cote, et al., 2003, Pulmonary gene expression profiling of inhaled ricin, Toxicon, v 41:813-22; Doebler, Wiltshire, et al., 1995, The distribution of [125I]ricin in mice following aerosol inhalation exposure, Toxicology, v 98:137-49) (Wilhelmsen and Pitt, 1996, Lesions of acute inhaled lethal ricin intoxication in rhesus monkeys, Veterinary Pathology, v 33:296-302), ricin inhalation results in respiratory distress and airway and pulmonary lesions. Inhalation of ricin in rats leads to rapid apoptotic changes in alveolar macrophages within 6 hours after exposure, finally culminating in inter alveolar edema at 12 and 15 h after exposure, mixed inflammatory cell infiltrates, alveolar flooding followed by tissue necrosis (Brown and White, 1997, Ultrastructure of rat lung following inhalation of ricin aerosol, International Journal of Experimental Pathology, v 78:267-76.). Compared to aerosol exposure, ricin administered orally is considerably less toxic (Balint, 1974, Ricin: the toxic protein of castor oil seeds, Toxicology, v 2:77-102). The literature indicates that the oral LD50 is 20 mg/kg, ˜1000 fold higher than the aerosol or systemic LDso values. Ingestion of whole castor beans results in severe abdominal pain, vomiting, diarrhea, and (depending on the number of beans and degree of mastication) death (Audi, Belson, et al., 2005, Ricin poisoning: a comprehensive review, JAMA, v 294:2342-51; Bradberry, Dickers, et al., 2003, Ricin poisoning, Toxicological Reviews, v 22:65-70; Mantis, 2005, Vaccines against the category B toxins: Staphylococcal enterotoxin B, epsilon toxin and ricin, Advanced Drug Delivery Reviews, v 57:1424-39; Olsnes, 2004, The history of ricin, abrin and related toxins, Toxicon, v 44:361-70). Rats challenged with ricin by gavage (1-30 mg/kg) developed dose-dependent lesions in the stomach and proximal small intestine (Sekine, Kawase, et al., 1986, Pathological study on mucosal changes in small intestine of rat by oral administration of ricin. I. Microscopical observation, Acta Pathol Jpn, v 36:1205-12), leading to widespread intestinal villus atrophy, crypt elongation, sloughing of the epithelium, and infiltration of inflammatory cells, including eosinophils and neutrophils (Sekine, Kawase, Nishimori, Mitarai, Harada, Ishiguro and Kikutani, 1986, Pathological study on mucosal changes in small intestine of rat by oral administration of ricin. I. Microscopical observation, Acta Pathol Jpn, v 36:1205-12; Ishiguro, Harada, et al., 1984, Effects of ricin, a protein toxin, on glucose absorption by rat small intestine. (Biochemical studies on oral toxicity of ricin. II), Chem Pharm Bull (Tokyo), v 32:3141-7; Ishiguro, Mitarai, et al., 1983, Biochemical studies on oral toxicity of ricin. I. Ricin administered orally can impair sugar absorption by rat small intestine, Chem Pharm Bull (Tokyo), v 31:3222-7; Ishiguro, Nakashima, et al., 1992, Interaction of toxic lectin ricin with epithelial cells of rat small intestine in vitro, Chem Pharm Bull (Tokyo), v 40:441-5; Ishiguro, Tanabe, et al., 1992, Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats, Journal of Pharmacobio-Dynamics, v 15:147-56). Similar histopathologic changes have been observed in mice challenged intragastrically with ricin (Yoder, Aslam, et al., 2007, Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication, Infection and Immunity, v 75:1745-50). It is postulated that following intestinal exposure, ricin ultimately disseminates from the mucosa into circulation (Ishiguro, Tanabe, Matori and Sakakibara, 1992, Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats, Journal of Pharmacobio-Dynamics, v 15:147-56), since ricin has been shown to cross polarized epithelial cell monolayers in vitro (Mantis, McGuinness, et al., 2006, Immunoglobulin A antibodies against ricin A and B subunits protect epithelial cells from ricin intoxication, Infection and Immunity, v 74:3455-62; van Deurs, Hansen, et al., 1990, Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium, Eur J Cell Biol, v 51:96-109). The inherent resistance of the gastrointestinal tract to ricin is likely due to a number of factors that impede toxin absorption, including intestinal proteases, digestive enzymes, mucus, and secretory IgA, whose galactose-rich oligosaccharide can competitively inhibit ricin attachment to the apical surfaces of intestinal epithelial cells (Mantis, Farrant, et al., 2004, Oligosaccharide side chains on human secretory IgA serve as receptors for ricin, Journal of Immunology, v 172:6838-45).
Efforts to create a vaccine against ricin have mostly focused on the RTA subunit. Two mutant versions of RTA have been extensively studied for their ability to promote antibody mediated immunity to ricin. One mutant, RVEc, completely removes the third folding domain, which mediates binding to RTB but is only targeted by non-neutralizing antibodies. A highly immunogenic loop in the first domain, which is also targeted only by non-neutralizing antibodies, is also removed. Another mutant, used in RiVax™, retains the entire structure of native RTA but contains two point mutations, Y80A and V76M, which completely remove both of RTA's known toxicities, ribotoxicity and vascular leak induction respectively. Studies in mice and rabbits demonstrated that RiVax™ is safe and immunogenic when administered by the intramuscular (i.m.) and intradermal (i.d.) routes. In a Phase I clinical trial, not all participants responded to RiVax™ when employed as an i.m. vaccine. In a second Phase I trial in which individuals received three i.m. immunizations over a span of 26 weeks, it was demonstrated that adsorption of RiVax™ to aluminum salt adjuvants enhanced RiVax™-specific serum antibodies (Ab) titers. The levels of anti-RiVax™ Ab were neither robust nor long lasting. Moreover, levels of toxin-neutralizing Ab were also extremely low in RiVax™-immunized individuals. Ricin neutralizing antibodies are likely the primary determinant of protective immunity to ricin. Collectively, these data defined the need to identify new adjuvants to boost the efficacy of RiVax™ and other antigens derived from ribosome inactivating proteins.
Anthrax is an acute infectious disease that is easily transmitted to humans by environmentally durable spores produced by gram positive bacterium Bacillus anthracis. Because the spores are robust and contagious, anthrax is considered a Category A bioterror threat. Anthrax infection can occur in three forms: cutaneous (skin), inhalation, and gastrointestinal. Inhaled spores can cause a rapidly progressing form of anthrax since the spores are transported to lymph nodes near the lungs where they germinate, releasing vegetative bacteria into the bloodstream. After infection in the bloodstream, the bacteria synthesize a complex series of toxin components that make up anthrax toxin, resulting in overwhelming toxemia that causes shock and organ failure.
The bacterium secretes 3 proteins which can combine to form two binary toxins, which cause the major pathology during anthrax infection of humans and animals. The cell binding component is Protective Antigen (PA), and once bound it is cleaved by a cell surface furin-like protease, leaving behind a 63 kDa fragment which then heptamerizes. The surface of the heptamer can bind up to 3 copies of edema factor (EF) and/or lethal factor (LF), forming edema toxin or lethal toxin, respectively. EF is an adenylate cyclase which increases intracellular cAMP concentrations. LF is a zinc-dependent metalloprotease which cleaves members of the MAPKK pathway, leading to apoptosis. Antibodies targeting PA can neutralize both toxins, and thus recombinant PA is the major antigen in the currently licensed anthrax vaccine adsorbed (AVA, Biothrax®). However, a six-dose vaccination series is required to sustain a high anti-PA IgG titers.
Treatment of anthrax involves long-term antibiotic therapy, since ungerminated spores can lie dormant in the lungs for up to 60 days. Only a few inhaled spores can cause inhalational anthrax poisoning. Once the toxin has entered the bloodstream, antibiotics are ineffective, and only toxin-specific therapy is effective. Passively transferred antibodies can neutralize anthrax toxins and can be used post-exposure in conjunction with antibiotics. Because of the long residence time of spores in the lung, it is possible to vaccinate post-exposure, but the onset of neutralizing antibodies must occur during the period of antibiotic therapy. Thus, there is a need for effective anthrax vaccines that can elicit rapid protective immunity. Moreover, there is a need for vaccine formulations that can be stored for extended periods prior to distribution, including vaccines intended to be used in “post-event” or “post-exposure” instances where vaccines would be deployed from the Strategic National Stockpile after weaponized use of hazardous biologicals. These products are currently stockpiled under environmentally controlled (cold chain) refrigerated storage conditions until those unpredictable events would occur, imposing huge logistical demands on maintaining the stockpile for those targeted interventions. Vaccines to be stored in the Strategic National Stockpile and used under emergency situations for biodefense are preferred to have long-term shelf life.
It is well accepted that PA is the dominant immunogen in AVA and target for protective immunity in pre-exposure and post exposure prophylaxis as a single component recombinant vaccine. Several aluminum adsorbed recombinant PA vaccines, based on expression of native PA in avirulent B. anthracis or E. coli, have been manufactured and tested in recent Phase I trials. Aluminum-adjuvant PA vaccines have been shown to be immunogenic in relationship to AVA, but it is difficult to directly state with the current evidence that an aluminum-adsorbed PA vaccine will in fact be the successor to AVA (Brown, Cox, et al., Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults, PLoS One, v 5:e13849; Campbell, Clement, et al., 2007, Safety, reactogenicity and immunogenicity of a recombinant protective antigen anthrax vaccine given to healthy adults, Hum Vaccin, v 3:205-11; Gorse, Keitel, et al., 2006, Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: a randomized, double-blinded, controlled, multicenter trial, Vaccine, v 24:5950-9). Limitations noted in the development of recombinant PA based vaccines have been rapid deamidation of rPA and loss of potency of liquid aluminum adsorbed vaccine. Therefore, the overall potency and related immunological correlates of effectiveness for any PA vaccine are still undeveloped. Improvements in the AVA vaccine have been directed at modifying the schedule of vaccination such that the current recommendation (by the FDA and the CDC) for AVA eliminated the requirement for a second vaccination at day 14 and changed the administration from subcutaneous to intramuscular. This first generation product is now approved for a vaccination regimen that involves injection by the intramuscular route at 0, 4 weeks, 6 months, and booster doses at 12 and 18 months (Marano, Plikaytis, et al., 2008, Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial, JAMA, v 300:1532-43). The changes were implemented following extensive study of AVA showing non inferiority of the 0, 4 i.m. regimen. The vaccine is routinely used in the USA for military personnel and has been acquired by HHS for the stockpile for civilian use in the event of a terrorist event and could be given upon Emergency Use Authorization (EUA) for post exposure prophylaxis (PEP) in conjunction with antibiotic therapy, prior to the time that FDA licensure is achieved for that indication. The ACIP has recommended the 0, 2, 4 week AVA subcutaneously for PEP along with a 60 day course of antibiotics. Using the currently licensed vaccination regimen with AVA, peak antibody titers are reached by week six through eight (after two injections), similar to peak titers reached using the former 0, 2, 4 week subcutaneous vaccination regimen (Marano, Plikaytis, Martin, Rose, Semenova, Martin, Freeman, Li, Mulligan, Parker, Babcock, Keitel, El Sahly, Poland, Jacobson, Keyserling, Soroka, Fox, Stamper, McNeil, Perkins, Messonnier and Quinn, 2008, Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial, JAMA, v 300:1532-43). Although antibodies decline until booster doses are given at 6, 12, and 18 months, it is presumed that protection against anthrax spore exposure has been achieved by the full regimen. Although no precise correlate of antibody-mediated immunity has been defined in non-human primate or rabbit models, protective titers in human subjects may be reached after two or three doses of AVA/Biothrax®.
A notable improvement in the AVA vaccine has been reported by adding immunostimulatory CpG sequences to AVA (Rynkiewicz, Rathkopf, et al., 2011, Marked enhancement of the immune response to BioThrax® (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers, Vaccine, v 29:6313-20; Klinman, Xie, et al., 2006, CpG oligonucleotides improve the protective immune response induced by the licensed anthrax vaccine, Ann N Y Acad Sci, v 1082:137-50; Klinman, Xie, et al., 2004, CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of Rhesus macaques, Vaccine, v 22:2881-6). Vaccination with modified product (AV7909) resulted in a demonstrably accelerated immune response in relationship to AVA achieving titers of TNA equivalent to peak AVA titers in approximately 21 days (after two doses of AVA7909 vaccine) (Rynkiewicz, Rathkopf, Sim, Waytes, Hopkins, Giri, DeMuria, Ransom, Quinn, Nabors and Nielsen, 2011, Marked enhancement of the immune response to BioThrax® (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers, Vaccine, v 29:6313-20). Further, peak titers of TNA were 6-8 fold higher using the AVA7909 vaccine, whereas at the same time AV7909 was associated with a higher frequency of injection site reactions. The modification of PA-based subunit vaccines with safe and effective adjuvants is thus encouraged by the results so far obtained with AV7909.
U.S. Pat. No. 7,037,503 (“Collier et al”) discloses dominant negative inhibitor (DNI) mutations of recombinant PA in which mutations of PA result in inhibition of its pore-forming ability, in which the mutations result in a PA molecule that is therapeutic for the treatment of toxemia caused by the secretion of anthrax toxins by bacteria circulating in the bloodstream of animals and humans afflicted with anthrax disease.
In an effort to improve upon anthrax vaccine, a dominant negative inhibitor (DNI) of PA has been developed. The anthrax DNI is an analog of rPA containing two mutations that prevent pore formation and translocation of the holotoxin in the cytosol. DNI binds to the same cell-surface receptors with the same affinity as PA and can form self-assembled heptamers on the surface of cells. The absence of the translocation step prevents DNI-dependent transport of Edema Factor and/or Lethal Factor into cell cytoplasm. Hence, complexes of DNI with LF or EF are nontoxic. DNI was originally proposed for use as an intravenous agent for post-aerosol exposure to anthrax spores to block holotoxin, since one subunit of DNI could block PA-dependent pore formation after heptamerization. It has been shown that animals vaccinated with the DNI antigen induced higher levels of antibodies to toxin and maintained high levels of protective antibody titers for up to one year without booster injections of antigen. The hyperimmunogencity of DNI has been attributed to the defects in pore formation which may enable more efficient antigen presentation in the context of class II MHC.
The anthrax DNI variant of recombinant PA was originally isolated by Collier et al. at Harvard in a screen for mutants of PA that were defective the steps of binding, transporting or internalization from the endosome (Sellman, Mourez, et al., 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v 292:695-7; Mourez, Kane, et al., 2001, Designing a polyvalent inhibitor of anthrax toxin, Nat Biotechnol, v 19:958-61). Mutated residues 397 and 425 in DNI are located in domain II, the pore-forming domain (Mourez, Yan, et al., 2003, Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis, Proc Natl Acad Sci USA, v 100:13803-8). Subsequent study revealed the substitution of DNI for one or more of the wild-type PAs within the heptamer blocked the movement of the toxin across endosomal membranes, effectively blocking the toxicity caused by liberation of LF and EF into the cell cytosol. It is hypothesized that these two mutations inhibit the essential conformational change of (PA63)7 from a ring-shaped core to a β barrel structure and thus prevent the heptamer from inserting itself into the endosomal membrane (Sellman, Nassi, et al., 2001, Point mutations in anthrax protective antigen that block translocation, J Biol Chem, v 276:8371-6). Consequently, these mutations inhibit the translocation of LF or EF to the cytosol and prevent cytotoxicity (Sellman, Mourez and Collier, 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v 292:695-7; Mourez, Kane, Mogridge, Metallo, Deschatelets, Sellman, Whitesides and Collier, 2001, Designing a polyvalent inhibitor of anthrax toxin, Nat Biotechnol, v 19:958-61; Sellman, Nassi and Collier, 2001, Point mutations in anthrax protective antigen that block translocation, J Biol Chem, v 276:8371-6). The DNI protein is activated normally, undergoes heptamerization, and binds LF and EF. The assembled complexes are endocytosed and trafficked to an acidic compartment similar to wild type PA. Upon exposure to acidic conditions, however, the complexes do not undergo membrane insertion or pore formation, and are not able to translocate the LF and EF into the cytosol. Thus, toxicity is completely blocked. The inactive, dead-end anthrax toxin complexes are believed to be trafficked to lysosomes and metabolized. DNI works in a dominant fashion meaning that DNI can be present in significantly lower concentrations than wt PA and still be effective in blocking toxin action. Studies performed in Fisher 344 rats subjected to challenge with LT (PA and LF) and administered DNI initially suggested a potential use of DNI in the treatment of patients infected with B. anthracis (Sellman, Mourez and Collier, 2001, Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax, Science, v 292:695-7). The DNI protein was developed on the basis that it could be used as a post exposure prophylaxis, and confirmatory studies were reported rats and then in rabbits with spore challenge. Subsequent to the rabbit and rat studies, several preliminary studies were conducted in mice and showed that the DNI mutant, with aluminum was more immunogenic than wild type PA (Aulinger, Roehrl, et al., 2005, Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines, Infect Immun, v 73:3408-14; Yan, Roehrl, et al., 2008, Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates, Vaccine, v 26:947-55). The DNI variant of PA induced 4-5 more anti-PA antibodies than the aluminum adsorbed PA vaccines. Increased immunogenicity could be due to that fact that the DNI PA does not escape the endosome and is more efficiently processed and presented to MHC molecules in antigen presenting cells.
In preclinical studies of PA or rPA vaccine, a spectrum of adjuvant formulations has been evaluated in relationship to aluminum-based adjuvants. A significant increase in toxin neutralizing antibody (TNA) titers in mice was obtained when CpG oligodeoxynucleotide adjuvant was added directly to AVA (Klinman, Klaschik, et al., 2010, Immunostimulatory CpG oligonucleotides: Effect on gene expression and utility as vaccine adjuvants, Vaccine, v 28:1919-23). These studies also demonstrated longer lasting TNA and increased protection from anthrax challenge when antibody levels had waned below the usually associated protective levels in mice, suggesting the induction of long-lasting memory B cells. Continuing Phase I studies have also demonstrated that CpG sequences with AVA induce higher TNA and ELISA antibodies resulting in a more rapid time to peak antibody levels. In addition to studies with aluminum adjuvants and CpG, studies conducted in the late 90s in Rhesus macaques indicated the saponin adjuvant, QS-21, and the partially detoxified lipid A (monophosphoryl lipid A/MPL) (Ivins, Pitt, et al., 1998, Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in Rhesus macaques, Vaccine, v 16:1141-8) could induce protection in comparison to unformulated rPA. Taken together, these data point to the need for additional adjuvants, and a need to control the factors that affect vaccine efficacy.
With respect to vaccines for anthrax and ricin, both of these vaccines are being developed individually and would be useful for military personnel and emergency first responders and in the event of dissemination of either biothreat agent during a biological attack. Thus it would be extremely useful if the vaccines could be administered simultaneously without compromising the response to either vaccine while still providing protection against whichever toxin might be encountered. There remains a need for a unique combination vaccine that is safe and effective against ricin and anthrax exposure and is capable of inducing neutralizing antibodies required for robust protection and vaccine efficacy. The combination will permit the concurrent immunization with fewer injections that would otherwise be needed with individual vaccine components.